Interpretive Summary: White blood cells produce certain small proteins which kill bacteria. We isolated one of these proteins called avian beta defensin-2 from the bone marrow of pheasant and quail and used various biochemical techniques to determine their structures. Understanding the structures of these biochemicals will help us developing methods to measure these in the blood of poultry to understand their resistance against infection.

Technical Abstract:
Pheasant and quail orthologues of avian ß-defensin 2 (AVBD2) were identified in methanol extracts of heterophil and bone marrow using matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). We used comparative pattern profiling before and after reduction/alkylation (RA) of the cell extracts with dithiothreitol (DTT)/2-iodoacetamide (IAA) to identify AvBD. Both heterophil and bone marrow extracts of pheasant and quail showed a single high intensity peak corresponding to m/z 4114 and 4162 respectively, which shifted by 348 Da upon carbamidomethylation (CAM) of 3 inherent disulfide bonds in AvBD. The corresponding intact and CAM AvBD2 peptides were purified by reverse phase HPLC. Partial sequences of the peptides were obtained by subjecting the purified CAM AvBD peptides to trypsin digestion followed by MALDI-MS and MALDI LIFT-TOF/TOF. The intact AvBD were also subjected to Edman degradation to obtain N-terminal sequences. Combining all of the above results and aligning the sequence stretches with other mature AvBD2, the pheasant and quail AvBD2 sequences were assembled. The intact and CAM peptides were also subjected to in source decay (ISD) to verify parts of the proposed sequence information. The proposed sequences of pheasant and quail, 'LFCKRGSCHFGRCPSHLIKVGSCFGFRSCCKWPWNA' and 'LFCRRGTCHFGNCPSDQIKVGNCFGFRSCCRWPWDA' showed high degree of similarities with other known AVBD2.